Abnormal vitamin D metabolism has been observed in both patients with x-linked hypophosphatemic rickets, as well as in the murine analog of this human disease, the C57BL6/J mouse with x-linked hypophosphatemia. In preliminary studies of cultured kidney cortical cells, cultured in serum free medium, from affected male mice (Hyp/y) and from their normal littermates (+/y), we have observed that the apparent basal activity of the renal 25-OH-D vitamin D3 1Alpha-hydroxylase is reduced in the Hyp/y cells as compared to the +/y cells. Enzyme activity is assessed by the conversion of 25-OH-D3 to 1,25-(OH)2-D3 with measurement of the latter by radioreceptor assay. We now propose to investigate the mechanisms which may account for the reduced basal enzyme activity of the 1Alpha-hydroxylase in the cultured Hyp/y cells. We will explore: 1) Whether the cultures of Hyp/y and of +/y cells contain approximately equivalent numbers of proximal tubular cells, as measured by Alpha-methylglucoside uptake, phlorizin binding, activity of other enzymes, and by ultrastructural appearance. 2) Whether the apparent difference in enzyme activity is present at lower and more physiological concentrations of 25-OH-D3. 3) Whether mitochondria isolated from Hyp/y cells also exhibit reduced 1Alpha-hydroxylase activity. 4) If mitochondrial enzyme activity is similar in Hyp/y and +/y cells, we will determine whether the reduced enzyme activity in the whole cells may be related to abnormalities in cell entry of 25-OH-D3 or whether Hyp/y cells contain a cytosolic inhibitor of mitochondrial enzyme activity. 5) In addition, since 1,25-(OH)2-D3 is known to regulate its own production, we will evaluate the possible role of alteration in 1,25-(OH)2-D3 binding to Hyp/y as compared to +/y cytoplasm. 6) To determine whether the reduced basal 1Alpha-hydroxylase activity in the Hyp/y cells persists when known stimuli of the enzyme are applied, we will evaluate the effects of parathyroid hormone and alterations in the phosphate concentration in culture media in Hyp/y as compared to +/y cells. We anticipate that these studies will provide new insights into the pathogenesis of abnormal regulation of renal vitamin D metabolism in x-linked hypophosphatemic rickets in mice. Such data are potentially relevant to a better understanding of the pathogenesis and treatment of hypophosphatemic rickets in human beings.